Immunocytochemical localization of alpha2,3(N)-sialyltransferase (ST3Gal III) in cell lines and rat kidney tissue sections: evidence for golgi and post-golgi localization.
Sialylation is a biosynthetic process occurring in the trans compartments of the Golgi apparatus. Corresponding evidence is based on localization and biochemical studies of alpha2, 6(N)-sialyltransferase (ST6Gal I) as previously reported. Here we describe generation and characterization of polyclonal antibodies to recombinant rat alpha2,3(N)-sialyltransferase (ST3Gal III) expressed as a soluble enzyme in Sf9 cells or as a beta-galactosidase-human-ST3Gal III fusion-protein from E.coli , respectively. These antibodies were used to localize ST3Gal III by immunofluorescence in various cell lines and rat kidney tissue sections. In transiently transfected COS cells the antibodies directed to soluble sialyltransferase or the sialyltransferase portion of the fusion-protein only recognized the recombinant antigen retained in the endoplasmic reticulum. However, an antibody fraction crossreactive with beta-galactosidase recognized natively expressed ST3Gal III which was found to be colocalized with beta1, 4-galactosyltransferase in the Golgi apparatus of several cultured cell lines. Antibodies affinity purified on the beta-galactosidase-ST3Gal III fusion-protein column derived from both antisera have then been used to localize the enzyme in perfusion-fixed rat kidney sections. We found strong staining of the Golgi apparatus of tubular epithelia and a brush-border-associated staining which colocalized with cytochemical staining of the H+ATPase. This subcellular localization was not observed for ST6Gal I which localized to the Golgi apparatus. These data show colocalization in the Golgi apparatus and different post-Golgi distributions of the two sialyltransferases.
Burger
P C
PC
Institute of Physiology, University of Zurich, Switzerland.
Lötscher
M
M
Streiff
M
M
Kleene
R
R
Kaissling
B
B
Berger
E G
EG
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000201:enzymology*; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000201:enzymology*; D007684:Kidney Tubules / Q000201:enzymology; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D012799:Sialyltransferases / Q000032:analysis*; D018411:Spodoptera / Q000235:genetics; D014162:Transfection / Q000235:genetics
D000906:Antibodies; D011994:Recombinant Proteins; D012799:Sialyltransferases